dimethyl 1,2-benzenedicarboxylate;reaction mass of: 1,2-dimethylpropylidene dihydroperoxide

Endpoint summary

Description of key information

In a 28 -day study with MIKP no adverse effects were observed up to the highest dose level of 75 mg/kg/day. In a 90 -day study with a structural analogue no adverse effects were observed up to 150 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2015-05-06 to 2015-12-xx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached justification in section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 51 - 54 days males and females
- Weight at study initiation: 180 - 239 g males, 117 - 145 g females
- Housing: 2 - 3 animals per cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water. ad libitum
- Acclimation period: 22 days for females, 85 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 75 mg/mL, 25 mg/mL and 10 mg/mL. Formulations were prepared in the formulation laboratory of Toxi-Coop Zrt. beforehand not longer than for three days and stored at 5 +/- 3°C until use.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle: A constant treatment volume of 2 mL dose preparation/kg body weight was administered.
- Lot/batch no.: 1410-5159, 1503-4337, 1503-4336, 1506-4604

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

MEKP content was determined in sunflower oil formulations using the previously validated reverse phase HPLC method with UV detection on a Kinetex 2.6u C18 100A column (100x4.6 mm). Detector: 205 nm.
MEKP concentrations in the samples varied in the range from 93% to 98 % in comparison to the nominal values.
Recovery of MEKP from sunflower oil formulations at two concentration levels (~10 and ~200 mg/mL) was 96 and 102 %.

Duration of treatment / exposure:

Duration of treatment: 90 or 91 days (depending on the day of necropsy)
Animals assigned to the recovery groups were treated identically up to and including Day 89 then they were observed for further four weeks.

Frequency of treatment:

Once a day on a 7 days/week basis, every day at a similar time (+/- 2 hours)

Remarks:

Doses / Concentrations:
0, 20, 50, 150 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose in the main study. Additionally, 5 animals per sex in the control and high dose group (recovery group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 150, 50 and 20 mg/kg bw/day was based on findings obtained in a previous repeated dose toxicity studies with MEKP in the Rat (28-day oral toxicity study in rats, Report no. SL-LT- 223/10; OECD 407; GLP).
- Post-exposure recovery period in satellite groups: Animals assigned to the recovery groups were treated identically up to and including Day 89 then they were observed for further four weeks.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter.
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed in the treatment period with an accuracy of 1 g on Day 0, then weekly. Individual body weight changes were calculated. Fasted body weight was measured on day of necropsy (Days 90 and 91 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION: Yes
The food consumption was determined in the treatment phase with an accuracy of 1 g on Day 7, then weekly by reweighing the non-consumed diet.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimation period, prior to test termination (Day 89)
- Dose groups that were examined: Repeated on all control and high dose test animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of recovery period (Day 118)
- Anaesthetic used for blood collection: Yes, under isofluran anesthesia
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of recovery period (Day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last exposure week (Day 86)
- Dose groups that were examined: all animals
- Battery of functions tested: Different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

ESTROUS CYCLE: Yes
- Time schedule for examinations: During the last two weeks of the treatment period (from Day 76 up to and including Day 90 or 91)
- Dose groups that were examined: all female animals
- Examinations: A vaginal smear was prepared from each female. After drying, smears were stained with 1 % aqueous methylene blue solution and were examined by a light microscope. The type of cycle (regular or irregular), number of days in pro-estrous, estrous and diestrous, number of cycle during the two weeks, number of animals with prolonged diestrus, number of animals with prolonged estrus were determined.

SPERM EXAMINATION
- Time for examinations: at Necropsy
- Quantitative examinations: The total number of homogenization of one side testis was enumerated. Testes and epididymides were frozen at the necropsy and enumeration was performed later.
- Qualitative examinations: Sperm motility was determined from ductus deferens at the necropsy. For the determination of the sperm motility the mean percentage of motile sperms was determined. The total sperm count and number of immotile sperms were recorded. Two samples were prepared from each animal.
A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No. 4), including organ weights
HISTOPATHOLOGY: Yes (see table No. 5)

Other examinations:

not applicable

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- estrous cycle
- hematology
- blood coagulation
- clinical chemistry
- organ weight data
- sperm parameters

The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.

Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.

The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings was calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male and one female animal died probably due to intra-tracheal applications of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male and one female animal died probably due to intra-tracheal applications of the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A slight and reversible elevation of the percentage of reticulocytes was observed in a dose related manner in male and female animals at 150 and 50 mg/kg bw/day.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased spleen weights (absolute and relative to body and brain weight) in male and females at 150 mg/kg bw/day and in females at 50 mg/kg bw/day.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Treatment period
Decreased activity and dyspnea were noted for one surviving male animal (1/14) at 150 mg/kg bw/day on Days 80 and 81. These slight and transient clinical signs were probably related to the test item effect.
Individual dermal alteration (alopecia) was observed on the shoulders in one male animal of 20 mg/kg bw/day group from Day 53 up to the end of the treatment period.

Recovery period
Clinical signs were not detected in the male or female animals of 150 mg/kg bw/day group. In one control female animal (1/5), alopecia was observed on the forelimbs from Day 115 up to the termination of the recovery period.
Alopecia is a common clinical sign in experimental rats of this age and was only present in single animal of the control and low dose group in this study

One male (1/15) and one female (1/15) animal was found dead at 150 mg/kg bw/day on Day 76 and Day 36, respectively.
In the male animal, decreased activity, dyspnea and cyanotic skin were observed between Day 74 and 76. Dark colored lungs and dark red liver were detected at the necropsy of this animal. Dyspnea was noted for the female animal one day before the death. Dark reddish mottled lungs, dark red liver and empty intestines were observed in this female animal at the necropsy. Histological examinations revealed acute catarrhal-purulent tracheitis accompanied with abundant fibrinous exudation into the lumen and diffuse congestion end edema in the lungs as probable cause of death of both animals. These lesions were originated from intra-tracheal application of the test item.

BODY WEIGHT AND WEIGHT GAIN
Treatment period
The body weight and body weight gain of the male and female animals were unaffected in all test item treated groups (150, 50 and 20 mg/kg bw/day) during the entire observation period. There were no statistically or biologically significant differences between the control and test item treated groups (male and female) at any dose level in the body weight.
Sporadic statistical significances were noted for the higher mean body weight gain of male animals at 150 and 50 mg/kg bw/day (between Days 49 and 56, between Days 70 and 77), as well as at 20 mg/kg bw/day (between Days 0 and 7) with respect to the control. The body weight gain was slightly lower than in the control group in male animals at 150 mg/kg bw/day during the last few days of the treatment period (between Days 84 and 89). There were no significant differences in the summarized mean body weight gain (between Days 0 and 89) of male animals between the control and test item treated groups.
Similarly, slight but statistically significant, higher mean body weight gain was observed in female animals at 50 mg/kg bw/day between Days 35 and 42, while the mean body weight gain was lower than in the control group in female animals at 150 mg/kg bw/day between Days 14 and 21 and at 150 and 50 mg/kg bw/day between Days 42 and 49.
These slight but statistically significant changes were considered to be without any toxicological relevance both in male and female animals.

Recovery period
The body weight and body weight gain were similar in the male and female animals of 150 mg/kg bw/day and control groups during the post-treatment (recovery) period. There were no statistically or biologically significant differences between the control and high dose recovery groups (male and female) in the body weight or body weight gain.

FOOD CONSUMPTION
Treatment period
The daily mean food consumption was comparable in the control and all test item treated groups (150, 50 or 20 mg/kg bw/day) during the entire observation period. Statistically or biologically significant differences between the control and test item treated groups (male and female) in the mean food consumption were not detected at any dose level.

Recovery period
There were no significant differences in the mean daily food consumption between the control and 150 mg/kg bw/day groups during the four weeks post-treatment period.

OPHTHALMOSCOPIC EXAMINATION
The eyes were without any detected abnormalities in all animals before treatment and in the control high dose group at termination of the treatment.

HAEMATOLOGY

Hematological investigations revealed slightly elevated percentages of reticulocytes in male and female animals at 150 and 50 mg/kg bw/day in a dose related manner. These slight changes were reversible as the difference in the percentages of reticulocytes between the control and high dose treated animals ceased up to the end of the recovery period. The slight elevation in the percentage of reticulocytes may be indicative of an increased rate of erythropoiesis. Although there were no signs of red blood cell destruction (hematology and clinical chemistry parameters, histopathology), the slightly higher spleen weight at the mid and high dose groups may refer to its increased function. However values remained well within the historical control ranges, therefore these changes were considered to be not adverse.

Main groups
At the termination of the treatment, statistical significances were noted in male animals for a slightly higher mean percentage of reticulocytes (RET) at 150 and 50 mg/kg bw/day, for a slightly higher mean red blood cell count (RBC) and for the higher mean hematocrit value (HCT) at 50 mg/kg bw/day with respect to the relevant control. In the female animals at 150 mg/kg bw/day, the mean hematocrit value (HCT), mean corpuscular (erythrocyte) volume (MCV) and mean percentage of reticulocytes were slightly higher while the mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) was slight lower than the appropriate control. Similarly, in female animals administered with 50 mg/kg bw/day, slight but statistically significant differences with respect to the control was detected at the higher mean corpuscular (erythrocyte) volume, higher mean hematocrit value and higher mean percentage of reticulocytes as well as at the slightly lower mean corpuscular (erythrocyte) hemoglobin concentration.

In the recovery group of 150 mg/kg bw/day dose, there were no statistically or biologically significant differences with respect to the control in the examined hematological parameters (male or female animals).

The statistical significant values (main and recovery groups) for RBC, HCT, MCV and MCHC were within the historical control ranges and were not related to doses (RBC, HCT ) therefore were not considered to be of biological or toxicological relevance.

CLINICAL CHEMISTRY

No pathologic test item effect was detected at the evaluation of clinical chemistry parameters at 150, 50 or 20 mg/kg bw/day.

Main groups
In male animals, statistically significant differences were noted for the lower mean concentration of chloride (Cl-) at 150 mg/kg bw/day and for the higher mean concentration of inorganic phosphorous (Pi) at 50 mg/kg bw/day.
In female animals, statistically significant differences with respect to the control were detected at 50 mg/kg bw/day (higher mean activity of aspartate aminotransferase – AST; and lower mean concentration of glucose – GLUC) and at 20 mg/kg bw/day (higher mean activity of aspartate aminotransferase and lower mean concentration of glucose and higher mean concentration of sodium – Na+).

Recovery groups
Statistical significances were observed in male animals of 150 mg/kg bw/day with respect to the control at the lower mean enzyme activity of aspartate aminotransferase and alanine aminotransferase (ALT) and at the slightly higher mean albumin: globulin ratio (A/G).
In female animals of 150 mg/kg bw/day recovery group, all examined clinical chemistry parameters corresponded with the relevant values of the control group.

These sporadic statistical differences (ALT, AST, GLUC, Pi, Cl-, Na+ and A/G) were considered to be of little or no biological relevance (main and recovery groups). Although the mentioned differences between the control and test item treated groups were statistically significant but were small

NEUROBEHAVIOUR

ORGAN WEIGHTS
Main groups
Slight, but statistically significant differences were detected in the higher mean spleen weights (absolute and relative to the body and brain weights) in male animals in 150 mg/kg bw/day group. Similar findings were also noted for spleen weight relative to body and brain weights of female animals at 150 mg/kg bw/day and for spleen weights (absolute and relative to body and brain weights) of female animals at 50 mg/kg bw/day with respect to the controls. The absolute mean spleen weight also exceeded the control value in female animals at 150 mg/kg bw/day however there was no statistically significance in this case. Spleen weight values remained within the historical control ranges, however along with changes in the percentages of reticulocytes a test item influence cannot be excluded.
The mean liver weight relative to the body weight was statistically significantly higher in female animals at 50 mg/kg bw/day group. The changes in liver weight remained well within the historical control ranges, hepatocellular damage were not detected at the histopathological examination and hematology investigations as well as clinical chemistry parameters did not revealed test item related abnormalities. Therefore liver weight changes were not considered to be toxicologically relevant.

Recovery groups
In the male animals of 150 mg/kg bw/day group, the mean spleen weight (absolute and relative to the body weight) and the testes weight (absolute and relative to brain weight) were slightly but statistically significantly higher than in the control group.
In the female animals of 150 mg/kg bw/day recovery group, statistical significances were noted for the higher mean liver weights and lower mean weights of ovaries (absolute and relative to body and brain weights, for both organs) with respect to the control.
These slight changes in the organ weights were considered to be of no toxicological relevance because the values were within the historical control ranges and there were no supporting findings in the examined clinical chemistry parameters or in histopathology.

GROSS PATHOLOGY
Dead animals
Necropsy findings of dead animals were indicative of congestion in the lungs and liver (dark colored or dark reddish mottled lungs, dark red liver) and referred to a circulatory disturbance developed during the death of these animals (1/1 male and 1/1 female at 150 mg/kg bw/day; on Days 76 and 36, respectively), which was also supported by the results of histopathological examination.

Main groups
Purulent abscess in the right side epididymidis (1/10), smaller than normal testes (1/10, both sides) and alopecia on the shoulders (1/10, both sides) were observed in single male animals at 20 mg/kg bw/day at the terminal necropsy. There were no macroscopic changes in male animals in the control, 50 or 150 mg/kg bw/day groups at termination of the treatment.
Alterations in the testes, epididymidis and dermal change were considered to be individual changes in single animals of the low dose (20 mg/kg bw/day) group (1/10, each).
Slight or moderate hydrometra was noted for several female animals in each group: 3/10 at 150 mg/kg bw/day, 1/10 at 50 mg/kg bw/day, 3/10 at 20 mg/kg bw/day and 3/10 in the control group at the end of the treatment period. Hydrometra related to the female sexual cycle is a frequent observation in experimental rats.

Recovery groups
There were no macroscopic changes in the male animals observed for four weeks after the termination of the treatment (control and 150 mg/kg bw/day).
In the female animals, slight or marked hydrometra (3/5 control; 2/4 at 150 mg/kg bw/day) were detected at the end of the recovery period.

HISTOPATHOLOGY
Dead animals
In the dead animals (1/15 male and 1/15 female at 150 g/kg bw/day, on Days 76 and 36, respectively), histological examination revealed acute catarrhal-purulent tracheitis accompanied with abundant fibrinous exudation into the lumen and diffuse congestion end edema in the lungs as probable cause of death. Moderate congestion in the liver was also noted for this dead male animal. These lesions were in connection with the mechanical procedure of treatment (erroneous intra tracheal application) and circulatory disturbance developed during the death of these animals. No degenerative or any other – possible toxic-lesions in the liver, kidney or other investigated organs were observed.

Main groups
In the lungs, alveolar emphysema was observed in one control (1/10) male animal, which was in connection with the hypoxia, dyspnoea and circulatory disturbance, developed during the exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/10 male control; 1/9 male at 150 mg/kg bw/day treated groups) was considered as a physiological, immuno-morphological phenomenon. Dilatation of uterine horns was detected in some female animals of control (3/10) and test item treated (3/10 at 150 mg/kg bw/day) groups at termination of the treatment and at the end of the recovery period (1/5 control and 1/4 at 150 mg/kg bw/day). Dilatation of uterine horns is a slight neuro-hormonal phenomenon in connection with the sexual function, the pro-estrus phase.
The focal inflammation in the epididymis (1/1 at 20 mg/kg bw/day) was considered as individual disease. The etiology of focal inflammation in the epididymis of rats (resemble “sperm granuloma”) is unknown, but experimentally it has been shown that they can occur as the result of reduced blood flow in the epididymis. (G.A. Boorman et al.).
In one male animal at 20 mg/kg bw/day (1/10), decreased intensity of spermatogenesis (both side) in the seminiferous tubuli of the testes occurred however the Sertoli cells, the spermatogonia and the spermatocytes were intact. Based on the incidence (only one animal was affected in the low dose group and all animals belonging to the high and middle dose group were negative) and the character of the lesion (no degeneration on the cytomorphology of remaining cell layers was observed), the finding was considered as individual phenomenon, probably in connection with decreased testosterone exposure (Vidal et al.).

Recovery groups
Hyperplasia of bronchus associated lymphoid tissue was also detected in male animals of the recovery groups (1/5 control and 1/5 at 150 mg/kg bw/day).
In one control female animal in the recovery group, focal alveolar histiocytosis was seen in the lungs (1/5), which was considered as an individual disease. The alveolar histiocytosis is a common incidental finding in rats, which consists of small collections of alveolar macrophages with abundant foamy (lipid-containing) cytoplasm.
Dilatation of uterine horns was also detected in single female animal at the end of the recovery period (1/5 control and 1/4 at 150 mg/kg bw/day).
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed.
The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.

SPERM EXAMINATIONS

Sperm examinations did not point out any test item related influence on the sperm cells at 150 mg/kg bw/day.
The total sperm count and sperms with not normal morphology (separated head and tail) were similar in the 150 mg/kg bw/day and in the control groups and not statistical significant. Statistical or biological significances were not detected at the mean percentage of motile sperm cells or mean percentage of immotile sperms in animals of 150 mg/kg bw/day.

EXAMINATION OF ESTROUS CYCLE

A test item influence on the estrous cycle was not detected.
The percent of animals with regular and irregular estrous cycle was similar in the control and test item treated groups. There were no significant differences between the control and test item treated groups in the mean number of cycles, days in pro-estrus, estrus or diestrus.

REFERENCES

G.A. Boorman, R.E.Chapin and K. Mitsumori: Testis and Epididymis Pp: 405-417. In: Pathology of the Fischer Rat. Reference and Atlas. Edited by Boorman, G. A., Montgomery, C. A and Mackenzie, W. F. Academic Press Inc. San Diego, New York, Boston, London, Sidney, Tokyo, Toronto, 1990

Vidal et al.: Reproductive System and Mammary Gland, pp 717-830. In: Toxicologic Pathology, Edited by Sahota et al: CRC Press, Taylor and Francis Group, 2013

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

not specified

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for Methyl-ethylketone peroxide (MEPK) was 150 mg/kg bw /day for male and females rats.

Executive summary:

The objective of this study was to obtain information on the possible health hazards likely to arise from repeated exposure with Methyl-ethylketone peroxide (MEKP) at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects. The test item was administered orally (by gavage) to Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 150, 50 and 20 mg/kg bw/day doses corresponding to concentrations of 0, 75, 25 and 10 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. 5 animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations).

The suitability of the chosen vehicle for the test item and sufficient stability of Methyl-ethylketone peroxide (MEKP) in the vehicle was analytically verified up front. Methyl-ethylketone peroxide (MEKP) was stable in the applied concentrations in sunflower oil at room temperature for 4 hours and in a refrigerator (5 +/-3°C) for 3 days. Concentrations of the test item in the dosing formulations varied from 93 % to 98 % of nominal concentrations at each analytical occasion, thereby confirming proper dosing. Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. Further sperm and estrous cycle examination were performed. The absolute and relative weights of selected organs were measured. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. In addition, epididymidis, testes and skin were also processed histologically in a single male animals of the 20 mg/kg bw/day dose group due to macroscopic observations at the necropsy. The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only.

Results:

One male and one female animal administered with 150 mg/kg bw/day died on Day 76 and 36 of the study, respectively. Decreased activity, dyspnea or cyanotic skin were noted for these animals 1-2 days before the death. In accordance with necropsy findings (dark colored or dark reddish mottled lungs, dark red liver), histological examinations revealed in both cases acute catarrhal-purulent tracheitis accompanied with abundant fibrinous exudation into the lumen and diffuse congestion and edema in the lungs as cause of the death. These lesions were probably due to intra-tracheal applications of the test item in both animals.Toxic signs related to the test item were not detected at any dose level (150, 50 or 20 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery.The body weight development of male and female animals wasnot affected by the test item.No test item related body weight, or body weight gain changes were observed with respect to controls at any dose level during the course of the study (150, 50 or 20 mg/kg bw/day).The daily mean food consumption was similar in animals of the control and test item treated groups (150, 50 and 20 mg/kg bw/day).There were noabnormalities in the eyes of animals in the high dose group at termination of the treatment (150 mg/kg bw/day).A slight and reversible elevation of the percentage of reticulocytes was observed in a dose related manner in male and female animals at 150 and 50 mg/kg bw/day at the termination of the treatment period. Although all values were lying well within the historical background range, the slight elevation correlated with the slight changes in the splenic weight, therefore a test item influence cannot be excluded.Clinical chemistryexaminations did not reveal test item related toxic changes in the evaluated parameters (150, 50 and 20 mg/kg bw/day).A test item influence on the estrous cycle was not detected (150, 50 and 20 mg/kg bw/day).Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 150 mg/kg bw/day dose.Specific macroscopic alterations related to treatment with the test item were not observed at the terminal necropsy or at the end of the recovery period.A test item influence cannot be excluded in changes of the weights of spleen (absolute and relative to body and brain weight) in male and female animals at 150 mg/kg bw/day and in female animals at 50 mg/kg bw/day. Although all values remained within the historical control ranges, along with the elevated percentage of reticulocytes a test item effect might be supposed.There were no histological lesions related to the test item effect.

Conclusion:

Based on these observations the no observed adverse effect level (NOAEL) for Methyl-ethylketone peroxide (MEPK) was 150 mg/kg bw /day for male and females rats.

 

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 February 2002 - 18 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Qualifier:

according to guideline

Guideline:

other: Substance Law 1987 (Japan) according to the notification of December 1986 by EA, Environmental Agency (no. 700), MHLW, Ministry of Health, Labor and Welfare (No. 1039) and METI, Ministry of Economy, Trade and Industry (No. 1014).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: males ± 244g, females ±177g
- Fasting period before study: NA
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type III, height 15 cm.) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived
- Water (e.g. ad libitum): Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: 04 February 2002 - 18 March 2002

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing (within 5 hours on day 11) and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance and vehicle. The formulations were stored at ambient temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX
- Concentration in vehicle: 0.589 and 14.5 mg/g propyleen glycol
- Amount of vehicle (if gavage): 5ml/kg, actual dose volumes were calculated weekly according to the latest body weight.
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purpose of the analytical study was to determine the accuracy of preparation, homogeneity and stability of the test substance in formulations.
From the results, it was concluded that for the formulations of Groups 2 - 4 the concentrations analysed were in agreement with target concentrations (93 % to 102 % of target based on peak 1 and 103 % to 104 % of target based on peak 2).
Based on the measurements, formulations of Groups 2 and 4 were considered to be homogeneous.
Formulations at target levels of 0.589 and 14.5 mg/g Trigonox R-938 in propylene glycol were found to be stable for at least 4 hours when stored at room temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
3, 15, 75 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 5 day range finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: 14-days
- Section schedule rationale (if not random): no data

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Twice daily animals were checked for mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily*, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena#. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate,
grade 3 = severe, grade 4 = very severe
* Not on day 2 of the recovery period.
# Not on day 28. Additionally on days 11 and 14 of the recovery phase.

BODY WEIGHT: Yes
- Time schedule for examinations: Treatment period: on days 1, 8, 15, 22 and 28. Recovery period: on days 1,8 and 14.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Subjective appraisal was maintained during the study, but no
quantitative investigation introduced as no effect was expected.

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes were examined following instillation of tropicamide solution (5 mg/ml): week 4 (all groups). Since no treatment-related ophthalmoscopic effects were noted in week 4, no ophthalmoscopic examination was performed in week 6.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination, between 7.30 and 9.30 a.m. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.25 ml), with citrate for clotting tests (1.0 ml) and Li-heparin treated tubes for clinical biochemistry parameters (1.0 ml).
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes; The animals were fasted overnight (with a maximum of approximately 22 hours) before blood sampling, but water was provided.
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination, between 7.30 and 9.30 a.m. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.25 ml), with citrate for clotting tests (1.0 ml) and Li-heparin treated tubes for clinical biochemistry parameters (1.0 ml).
- Animals fasted: Yes; The animals were fasted overnight (with a maximum of approximately 22 hours) before blood sampling, but water was provided.
- How many animals:all
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
During week 4 of treatment, the following tests were performed on all animals:
hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent). motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

Sacrifice and pathology:

NECROPSY
All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a 4% formaldehyde solution:

Identification marks: not processed, Adrenal glands, Aorta, Brain - representative regions, including cerebrum, cerebellum and pons., Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur inclUding joint), Heart, Ileum, Ovaries, Pancreas, Peyer's patches Uejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin
Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
The following slides were examined by a pathologist: all tissues and organs collected at the scheduled sacrifice from all main group animals of the control and the highest dose group (tissues mentioned within brackets were not examined as these did not show signs of toxicity or target organ involvement); all tissues and organs from all animals of all dose groups which died spontaneously; all gross lesions of all animals (all dose groups). Based on the treatment-related morphologic changes found in organs of animals in the high dose group, liver, stomach and spleen from all rats of the intermediate dose groups and recovery groups were also examined. All abnormalities were described and included in the report.

Other examinations:

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Epididymides, Kidneys, Ovaries, Testes, Brain, Heart, Liver, Spleen, Thymus

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet
display different test statistics values.

References:
- C.W. Dunnett, Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50,1096-1121 (1955).
- R.G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981).
- R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

low-grade cytoplasmatic vacuolation of the perilobular hepatocytes was noted for all high dose females at the end of treatment. This finding had resolved after a 14day treatment free recovery period and the effect is considered not adverse.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment-related mortality occurred during the study. Two females (nos. 40 and 56 of the control and 75 mg/kg/day dose group, respectively) had died at blood sampling at the end of the recovery phase. These were considered accidental deaths.
There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. Incidental findings that were noted included salivation, rales, piloerection, alopecia, scabs and brown staining of the fur. These findings are more often noted in rats of this age and strain that are housed and treated under the conditions in this study. At the incidence observed, these signs were considered of no toxicological significance. No clinical signs were present among group 2 males and females and group 4 males.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.
Reduced body weights among animals of the control and high dose groups on day 1 of recovery were due to fasting prior to blood sampling. Minimal weight loss was measured for one control female (no. 21) on day 28. This incidental occurrence did not affect the interpretation of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no treatment-related differences in food consumption before or after allowance for body weight between treated and control animals.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.
Statistically significant differences in white blood cell counts between control and high dose animals at the end of the recovery occurred in the absence of pathological support and of a similar response at the end of treatment. The apparent reduction of white blood cell counts and haemoglobin values among group 2 and 3 males respectively, were considered to have arisen by chance and occurred in the absence of a dose-related response. These changes were therefore considered to be of no toxicological relevance.

CLINICAL CHEMISTRY
There were no differences noted between control and treated rats that were considered to berelated to treatment.
The reduction of aspartate aminotransferase activity of high dose females was slight and the opposite effect would be expected in case of organ toxicity. The statistically significant increase of the mean glucose value of group 3 males was considered to be caused by increased individual values of two males (nos. 17 and 18). Other changes of means of intermediate dose groups attaining statistical significance were not dose-related and values were considered to be normal. The reduced mean calcium level of high dose males and reduced urea values of females dosed at 15 or 75 mg/kg/day at the end of treatment remained comparable to values of similar studies. Changes attaining statistical significance at the end of recovery were absent at the end of treatment. These findings were considered to be of no toxicological significance.

NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the test animals, when compared to control animals.
The variation in motor activity was not related to the dose and did not indicate a relation with treatment.

ORGAN WEIGHTS
Organ weights and organ:body weight ratios of treated animals were considered not to have been affected by treatment.
Statistically significant changes in heart, kidney and liver weights among treated males at the end of treatment occurred in the absence of a dose-related response and/or were absent when corrected for body weight. These changes were therefore considered not to be a sign of toxicity.

GROSS PATHOLOGY
There were no treatment-related macroscopic abnormalities noted. An enlarged spleen in one male and one female of the high dose group and in one male dosed
at 15 mg/kg at the end of treatment were not supported by a treatment-related microscopic lesion. Other incidental findings noted among control and/or treated animals are occasionally seen among rats used in these types of studies. These were considered changes of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
An increased incidence of low-grade cytoplasmatic vacuolation of the perilobular hepatocytes was noted for all high dose females at the end of treatment. This finding had resolved after a 14day treatment free recovery period and were considered as not adverse.
All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats. No treatment-related microscopic lesion was found for the enlarged spleen in one male and one female of the high dose group and in one male dosed at 15 mg/kg at the end of treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Analysis of Dose Preparations

Analyses were based on the presence of two peaks in the HPLC chromatogram. Formulations in propylene glycol were noted as stable for at least 4 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 93 % to 104 % of nominal (based on two peaks), which was considered to represent an acceptable level of accuracy for formulations of this type.

Conclusions:

Wistar rats were treated by oral gavage for 28 consecutive days at dose levels up to 75 mg/kg/day.
Treatment resulted in minor effects noted at microscopic examination, i.e. an increase of cytoplasmatic vacuolation in the liver of all high dose females. This finding appeared reversible during a 14-day treatment-free period.
There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination and organ weight determination that were considered to be an effect of treatment.
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for MIKP of 75 mg/kg/day was established.

Executive summary:

Subacute 28-day oral toxicity with MIPKP by daily gavage in the rat, followed by a 14 day recovery period.

Report summary 2002

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0,3, 15 and 75 mg/kg/day.

The study was based on the following guidelines:

- EC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996.

- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995.

- Japanese Substance Law 1987, Notification of Dec. 1986 by EA, MHLW and MET!.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats (Crl:(WI) BR). One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; ophthalmoscopy at week 4; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Formulations: Accuracy, homogeneity and stability over 4 hours of formulations of test substance in propylene glycol were demonstrated by analyses.

Treatment-related findings observed were as follows:

3 mg/kg/day:- No treatment-related findings noted.

15 mg/kg/day:- No treatment-related findings noted.

75 mg/kg/day:- Increased incidence of low-grade cytoplasmatic vacuolation of the perilobular hepatocytes in all females, which had resolved at the end of recovery.

Treatment resulted in minor effects noted at microscopic examination, i.e. an increase of cytoplasmatic vacuolation in the liver of all high dose females. This finding appeared reversible during a 14-day treatment-free period.

There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination and organ weight determination that were considered to be an effect of treatment.

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for MIPKP of 15 mg/kg/day was established.

2018 expert statement

In this 28-day oral toxicity study, treatment resulted in minor histopathological effects consisting of cytoplasmic

vacuolation in perilobular (periportal) hepatocytes of all (5/5) high dose (75 mg/kg/day) females. This finding was reversible during the 14-day recovery period.

No other indication of toxicity were noted in the parameters determined in this study. In the histopathology report (compiled by Principal Investigator Dr. M. Martland, Pre-

clinical Safety Cons ultants Ltd .) this hepatic lesion was judged to be adverse. As such, the NOAEL in this study was set at 15 mg/kg/day.

This NOAEL was re-addressed in the light of current insights on interpretation of adverse findings (Kerlin et al, 2016).

It was judged that based on the following arguments, the cytoplasmic vacuolation in the liver may not be considered adverse:

• Severity was of a low grade (minimal to slight), and was not accompanied by any other treatment-related liver lesions.

• The finding was reversible within 14 days after cessation of treatment, i.e. this was not a permanent lesion.

• Liver weights were not considered to be affected by treatment.

• There were no clinical pathology findings that would be indicative of liver dysfunction.

• There were no adverse findings in any of the other (in-life) parameters.

The pathologist reported that: “ this finding is not an unusual finding but was apparent at higher incidence than w ould be expected for a spontaneous lesion

”). This finding is therefore considered to be related to treatment. However, b ased on the above considerations, this would not be judged as “adverse” in the context of this study

and based on current insights on interpretation of adverse findings

In conclusion, the NOAEL may be set to the highest dose level tested in this study, i.e. 75 mg/kg/day.

Kerlin, R., Bolon, B., Burkhardt, J., Francke, S., Greaves, P., Meador, V., Popp, P. (2016). Scientific and Regulatory Policy Committee: Recommended (“Best”) Practice for

Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies. Toxicol. Pathol. 44(2), 147-

162.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

K1 study, OECD guideline and GLP

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a 28 -day study with MIKP no adverse effects were observed up to the highest dose level of 75 mg/kg/day. In a 90 -day study with a structural analogue no adverse effects were observed up to 150 mg/kg bw/d. Based on these findings MIKP is not classified for repeated dose toxicity.